Lysophosphatidic acid acetyltransferases

ABSTRACT

An isolated nucleic acid fragment encoding an LPAAT isozyme is disclosed. Construction of a chimeric gene encoding all or a portion of the LPAAT isozyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the LPAAT isozyme in a transformed host cell is also disclosed.

This application is a divisional of U.S. patent application Ser. No.11/009,658 filed Dec. 10, 2004, issued as U.S. Pat. No. 7,235,714, whichis a divisional of U.S. patent application Ser. No. 09/914,098 filedAug. 22, 2001, issued as U.S. Pat. No. 6,855,863, which is a nationalstage filing of PCT Application No. PCT/US00/04526 filed Feb. 22, 2000,now abandoned, which claims the benefit of U.S. Provisional ApplicationNo. 60/121,119, filed Feb. 22, 1999. The entire content of each of theseSpecifications is incorporated herein by reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology and, inparticular, this invention pertains to isolated polynucleotides encodinglysophosphatidic acid acyltransferases in plants and seeds.

BACKGROUND OF THE INVENTION

Triacylglycerols are nonpolar, water-insoluble fatty acid triesters ofglycerols. Triacylglycerols differ according to the identity andplacement of their three fatty acid residues. Lysophosphatidic acidacyltransferase (EC 2.3.1.51), also called1-acyl-sn-glycerol-3-phosphate acyltransferase, 1-AGP acyltransferase,1-AGPAT, lysophosphatidic acid transferase, and LPAAT, catalyzes theattachment of the second acyl group to the glycerol backbone duringde-novo biosynthesis of triacylglycerols.

The fatty acid distribution in triacylglycerols is thought to bedependent on the specificities of the acyltransferases involved in theirbiosynthesis. Although no plant LPAAT has been purified to completion,spinach leaves have at least two systems which reside in differentsubcellular compartments (chloroplast inner membrane and the endoplasmicreticulum) and which incorporate different fatty acids into the glycerolbackbone (Frentzen et al. (1984) in Structure, function and metabolismof plant lipids; Siegenthaler and Eichenberger, eds. pp 105-110).Isolation of LPAAT genes from Limnanthes douglasii is dependent on theapproach used to isolate the clone. Two different clones have beenisolated which varied in their expression patterns, in their ability tocomplement an E. coli temperature-sensitive mutant defective in LPAATactivity and in their ability to hybridize to the already known maizeLPAAT (Brown et al. (1995) Plant Mol. Biol. 29:267-278). Thus, thepresence of many other LPAATs with different specificities, subcellularlocations and activities is expected.

Production of industrially-significant oils in seed oil plants has beena quest of the agricultural industry of some time now. Introduction ofthe yeast LPAAT sequence into Arabidopsis and B. napus results inincreased seed oil content in many transgenic plants and in changes inseed oil composition (Zou et al. (1997) Plant Cell 9:909-923).

SUMMARY OF THE INVENTION

The invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 100 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, and 52, or (b) a second nucleotide sequencecomprising the complement of the first nucleotide sequence.

In a second embodiment, it is preferred that the isolated polynucleotideof the claimed invention comprises a first nucleotide sequence whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, and 51, that codes for the polypeptide selectedfrom the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.

In a third embodiment, this invention concerns a chimeric genecomprising an isolated polynucleotide of the present invention operablylinked to suitable regulatory sequences.

In a fourth embodiment, this invention concerns an isolated host cellcomprising a chimeric gene of the present invention or an isolatedpolynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

In a fifth embodiment, the present invention concerns a process forproducing an isolated host cell comprising a chimeric gene of thepresent invention or an isolated polynucleotide of the presentinvention, the process comprising either transforming or transfecting anisolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the present invention.

In a sixth embodiment, the invention also relates to lysophosphatidicacid acyltransferase (LPAAT isozymes) polypeptides of at least 100 aminoacids comprising at least 80% homology based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, and 52.

In a seventh embodiment, the invention concerns a method of selecting anisolated polynucleotide that affects the level of expression of an LPAATisozyme polypeptide in a host cell, preferably a plant cell, the methodcomprising the steps of: (a) constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene of the presentinvention; (b) introducing the isolated polynucleotide or the isolatedchimeric gene into a host cell; (c) measuring the level the LPAATisozyme polypeptide in the host cell containing the isolatedpolynucleotide; and (d) comparing the level of the LPAAT isozymepolypeptide in the host cell containing the isolated polynucleotide withthe level of the LPAAT isozyme polypeptide in the host cell that doesnot contain the isolated polynucleotide.

In an eighth embodiment, the invention concerns a method of obtaining anucleic acid fragment encoding a substantial portion of an LPAAT isozymepolypeptide, preferably a plant LPAAT isozyme polypeptide, comprisingthe steps of: synthesizing an oligonucleotide primer comprising anucleotide sequence of at least 60 (preferably at least 40, mostpreferably at least 30) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and51 and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a portion of an LPAAT isozyme amino acidsequence.

In a ninth embodiment, the invention concerns a method of obtaining anucleic acid fragment encoding all or a substantial portion of the aminoacid sequence encoding an LPAAT isozyme polypeptide comprising the stepsof: probing a cDNA or genomic library with an isolated polynucleotide ofthe present invention; identifying a DNA clone that hybridizes with anisolated polynucleotide of the present invention; isolating theidentified DNA clone; and sequencing the cDNA or genomic fragment thatcomprises the isolated DNA clone.

In a tenth embodiment, this invention concerns a composition, such as ahybridization mixture, comprising an isolated polynucleotide of thepresent invention.

In an eleventh embodiment, this invention concerns an isolatedpolynucleotide of the present invention comprising at least 30contiguous nucleotides derived from a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51.

In a twelfth embodiment, this invention concerns a method for positiveselection of a transformed cell comprising: (a) transforming a host cellwith the chimeric gene of the present invention or an expressioncassette of the present invention; and (b) growing the transformed hostcell, preferably plant cell, such as a monocot or a dicot, underconditions which allow expression of the LPAAT isozyme polynucleotide inan amount sufficient to complement a null mutant to provide a positiveselection means.

In a thirteenth embodiment, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) first nucleotide sequence encoding a polypeptide ofat least 100 amino acids having at least 95% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:20, 22, 24, 54, 56, and 58 or (b) asecond nucleotide sequence comprising the complement of the firstnucleotide sequence. All of the embodiments described above areapplicable with the exception of the particular sequences involved andthe sequence identity being at least 95% as noted in the appropriateclaims.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying Sequence Listing which form a part ofthis application.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Lysophosphatidic Acid Acyltransferases SEQ ID NO: Protein CloneDesignation (Nucleotide) (Amino Acid) Corn Polypeptide Similar to Contigof: 1 2 Mus musculus LPAAT p0018.chssd06r p0104.cabbd29r cca.pk0027.c9p0018.chstw94r p0094.csssl20r Soybean Polypeptide Similar tosl2.pk121.a19 3 4 Mus musculus LPAAT Wheat Polypeptide Similar to Contigof: 5 6 Mus musculus LPAAT wlm1.pk0018.g6 wre1n.pk0040.h11wre1n.pk0064.g7 Corn Polypeptide Similar to Contig of: 7 8 B.pseudomallei LPAAT ceb5.pk0049.b3 cen3n.pk0027.f6 Soybean PolypeptideSimilar to sgs1c.pk001.i16 9 10 B. pseudomallei LPAAT Wheat PolypeptideSimilar to wre1n.pk0027.d4 11 12 B. pseudomallei LPAAT ArabidopsisPolypeptide Similar ads1c.pk005.i10 13 14 to Arabidopsis thalianaProtein Rice Polypeptide Similar to Contig of: 15 16 Arabidopsisthaliana Protein rls6.pk0076.d5 rlr24.pk0068.e3 Soybean PolypeptideSimilar to scb1c.pk003.d18 17 18 Arabidopsis thaliana Protein RicePolypeptide Similar to Contig of: 19 20 Corn LPAAT rr1.pk0004.a10rr1.pk0039.e10 Soybean Polypeptide Similar to Contig of: 21 22 CornLPAAT se4.cp0008.b2 sl2.pk0033.c1 Wheat Polypeptide Similar to Contigof: 23 24 Corn LPAAT wlk1.pk0004.e7 wle1n.pk0002.g3 Catalpa PolypeptideSimilar to ncs.pk0013.d2:fis 25 26 Mus musculus LPAAT Corn PolypeptideSimilar to Contig of: 27 28 Mus musculus LPAAT ceb1.pk0011.d11ceb5.pk0053.e3 p0010.cbpbq45r p0018.chssd06r:fis Rice PolypeptideSimilar to rlr2.pk0028.d6:fis 29 30 Mus musculus LPAAT SorghumPolypeptide Similar to gds1c.pk002.a19:fis 31 32 Mus musculus LPAATSoybean Polypeptide Similar to sl2.pk121.a19:fis 33 34 Mus musculusLPAAT Catalpa Polypeptide Similar to ncs.pk0009.f12:fis 35 36 B.pseudomallei LPAAT Wheat Polypeptide Similar to wre1n.pk0027.d4:fis 3738 B. pseudomallei LPAAT Corn Polypeptide Similar to Contig of: 39 40Arabidopsis thaliana Protein ceb1.mn0001.d12:fis cpe1c.pk006.e1 RicePolypeptide Similar to rls6.pk0076.d5:fis 41 42 A. thaliana ProteinSoybean Polypeptide Similar to scb1c.pk003.d18:fis 43 44 Arabidopsisthaliana Protein Corn Polypeptide Similar to cco1n.pk062.p19 45 46 A.thaliana acyltransferase Rice Polypeptide Similar to rlr6.pk0094.f6:fis47 48 A. thaliana acyltransferase Soybean Polypeptide Similar tosdp4c.pk006.n11:fis 49 50 A. thaliana acyltransferase SoybeanPolypeptide Similar to Contig of: 51 52 A. thaliana acyltransferasesgs1c.pk005.k7 sgs5c.pk0003.e7 Rice Polypeptide Similar torr1.pk0004.a10:fis 53 54 Corn LPAAT Soybean Polypeptide Similar tosl2.pk0033.c1:fis 55 56 Corn LPAAT Wheat Polypeptide Similar towlk1.pk0004.e7:fis 57 58 Corn LPAAT

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide” and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 60contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 30 contiguous nucleotides derived from anucleotide sequence selected from the group consisting of (a) SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, and 51, or the complement of such sequences and /or (b)SEQ ID NOs:19, 21, 23, 53, 55, and 57 or the complement of suchsequences. The term “isolated” polynucleotide is one that has beensubstantially separated or purified away from other nucleic acidsequences in the cell of the organism in which the nucleic acidnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, by conventional nucleic acid purification methods. The term alsoembraces recombinant polynucleotides and chemically synthesizedpolynucleotides.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides derived from the instantnucleic acid fragment can be constructed and introduced into a plant orplant cell. The level of the polypeptide encoded by the unmodifiednucleic acid fragment present in a plant or plant cell exposed to thesubstantially similar nucleic fragment can then be compared to the levelof the polypeptide in a plant or plant cell that is not exposed to thesubstantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 60 (preferably at least 40, mostpreferably at least 30) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of (a) SEQ ID NOs:1, 3, 5,7, 9, 11, 13, 15, 17, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, and 51 and the complement of such nucleotide sequences, and/or (b)SEQ ID NOs:19, 21, 23, 53, 55, and 57 and the complement of suchnucleotide sequences may be used in methods of selecting an isolatedpolynucleotide that affects the expression of an LPAAT isozymepolypeptide in a host cell. A method of selecting an isolatedpolynucleotide that affects the level of expression of a polypeptide ina host cell (eukaryotic, such as plant or yeast, prokaryotic such asbacterial, or viral) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or theisolated chimeric gene into a host cell; measuring the level apolypeptide in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide in the host cell containing theisolated polynucleotide with the level of a polypeptide in a host cellthat does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above homologies but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, asequence of ten or more contiguous amino acids or thirty or morecontiguous nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a DNA that is complementary toand derived from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double strandedform using, for example, the klenow fragment of DNA polymerase I.“Sense” RNA refers to an RNA transcript that includes the mRNA and socan be translated into a polypeptide by the cell. “Antisense RNA” refersto an RNA transcript that is complementary to all or part of a targetprimary transcript or mRNA and that blocks the expression of a targetgene (see U.S. Pat. No. 5,107,065, incorporated herein by reference).The complementarity of an antisense RNA may be with any part of thespecific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that maynot be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation.

The term “recombinant” means, for example, that a recombinant nucleicacid sequence is made by an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is a technique for the synthesis oflarge quantities of specific DNA segments. It consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwark, Conn.).Typically, the double-stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) firstnucleotide sequence encoding a polypeptide of at least 100 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, and 52, or (b) a second nucleotide sequencecomprising the complement of the first nucleotide sequence.

Preferably, the first nucleotide sequence comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and51, that codes for the polypeptide selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, and 52.

The present invention also concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) first nucleotide sequence encoding a polypeptide of at least 100amino acids having at least 95% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:20, 22, 24, 54, 56, and 58 or (b) a secondnucleotide sequence comprising the complement of the first nucleotidesequence.

Preferably, the first nucleotide sequence comprises a nucleic acidsequence selected from the group consisting of SEQ ID Nos:19, 21, 23,53, 55 and 57 that codes for the polypeptide selected from the groupconsisting of SEQ ID NOs:20, 22, 24, 54, 56, and 58.

Nucleic acid fragments encoding at least a portion of several LPAATisozymes have been isolated and identified by comparison of random plantcDNA sequences to public databases containing nucleotide and proteinsequences using the BLAST algorithms well known to those skilled in theart. The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other LPAAT isozymes, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, the entire sequences canbe used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 60 (preferably at least 40, most preferably at least 30)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of:

-   -   (a) SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 25, 27, 29, 31,        33, 35, 37, 39, 41, 43, 45, 47, 49, and 51 and the complement of        such nucleotide sequences may be used in such methods to obtain        a nucleic acid fragment encoding a substantial portion of an        amino acid sequence of a polypeptide, and/or    -   (b) SEQ ID NOs:19, 21, 23, 53, 55 and 57 and the complement of        such nucleotide sequences may be used in such methods to obtain        a nucleic acid fragment encoding a substantial portion of an        amino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of an LPAAT isozyme polypeptidepreferably a substantial portion of a plant LPAAT isozyme polypeptide,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least 60 (preferably at least 40,most preferably at least 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of:

-   -   (a) SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 25, 27, 29, 31,        33, 35, 37, 39, 41, 43, 45, 47, 49, and 51 and the complement of        such nucleotide sequences; and/or    -   (b) SEQ ID NOs:19, 21, 23, 53, 55 and 57 and the complement of        such nucleotide sequences,        and amplifying a nucleic acid fragment (preferably a cDNA        inserted in a cloning vector) using the oligonucleotide primer.        The amplified nucleic acid fragment preferably will encode a        portion of an LPAAT isozyme polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns host cells comprisingeither the chimeric genes of the invention as described herein or anisolated polynucleotide of the invention as described herein. Examplesof host cells which can be used to practice the invention include, butare not limited to, yeast, bacteria, plants, and viruses.

As was noted above, the nucleic acid polynucleotides of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of specifictriacylglycerols in those cells. For example overexpression of an LPAATsimilar to the maize LPAAT, such as those contained in Example 6, willresult in higher oil content in the seed, stem and leaf whileoverexpression of LPAAT similar to Burkholderia pseudomallei will resultin larger accumulation of oil in seed.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the isolated polynucleotide (or chimericgene) may be constructed. The choice of plasmid vector is dependent uponthe method that will be used to transform host plants. The skilledartisan is well aware of the genetic elements that must be present onthe plasmid vector in order to successfully transform, select andpropagate host cells containing the chimeric gene. The skilled artisanwill also recognize that different independent transformation eventswill result in different levels and patterns of expression (Jones et al.(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics218:78-86), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns an polypeptide ofat least 100 amino acids that has at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.

In still another embodiment, the present invention also concerns apolypeptide of at least 100 amino acids that has at least 95% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:20, 22, 24, 54, 56, and58.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded LPAAT isozyme. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 9).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and as markers for traitslinked to those genes. Such information may be useful in plant breedingin order to develop lines with desired phenotypes. For example, theinstant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth above is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries, Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various Arabidopsis, catalpa,corn, rice, sorghum, soybean, and wheat tissues were prepared. Thecharacteristics of the libraries are described below.

TABLE 2 cDNA Libraries from Arabidopsis, Catalpa, Corn, Rice, Sorghum,Soybean, and Wheat Library Tissue Clone ads1c Arabidopsis Wassilewskija6 day old seedlings ads1c.pk005.i10 cca Corn Callus Type II Tissue,Undifferentiated, Highly cca.pk0027.c9 Transformable cco1n Corn Cob of67 Day Old Plants Grown in Green House¹ cco1n.pk062.p19:fis ceb1 CornEmbryo 10 to 11 Days After Pollination ceb1.mn0001.d12:fis ceb1 CornEmbryo 10 to 11 Days After Pollination ceb1.pk0011.d11 ceb5 Corn Embryo30 Days After Pollination ceb5.pk0049.b3 ceb5 Corn Embryo 30 Days AfterPollination ceb5.pk0053.e3 cen3n Corn Endosperm 20 Days AfterPollination¹ cen3n.pk0027.f6 cpe1c Corn pooled BMS treated withchemicals related to cpe1c.pk006.e1 phosphatase² gds1c Sorghum Seed 20Days After Pollination gds1c.pk002.a19:fis ncs Catalpa speciosaDeveloping Seed ncs.pk0009.f12:fis ncs Catalpa speciosa Developing Seedncs.pk0013.d2:fis p0010 Corn Log Phase Suspension Cells Treated WithA23187³ p0010.cbpbq45r to Induce Mass Apoptosis p0018 Corn SeedlingAfter 10 Day Drought, Heat Shocked for p0018.chssd06r 24 Hours,Harvested After Recovery at Normal Growth Conditions for 8 Hours p0018Corn Seedling After 10 Day Drought, Heat Shocked for p0018.chstw94r 24Hours, Harvested After Recovery at Normal Growth Conditions for 8 Hoursp0094 Corn Leaf Collars for the Ear Leaf (EL) and the Next Leafp0094.csssl20r Above and Below the EL¹ p0104 Corn Roots V5 Stage⁴, CornRoot Worm Infested¹ p0104.cabbd29r rlr2 Rice Leaf 15 Days AfterGermination, 2 Hours After rlr2.pk0028.d6:fis Infection of StrainMagaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant rlr24 Rice Leaf 15Days After Germination, 24 Hours After rlr24.pk0068.e3 Infection ofStrain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant rlr6 Rice Leaf15 Days After Germination, 6 Hours After rlr6.pk0094.f6:fis Infection ofStrain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant rls6 Rice Leaf15 Days After Germination, 6 Hours After rls6.pk0076.d5 Infection ofStrain Magaporthe grisea 4360-R-67 (AVR2-YAMO); Susceptible rr1 RiceRoot of Two Week Old Developing Seedling rr1.pk0004.a10 rr1 Rice Root ofTwo Week Old Developing Seedling rr1.pk0039.e10 scb1c SoybeanEmbryogenic Suspension Culture Subjected to scb1c.pk003.d18 4Bombardments and Collected 12 Hours Later sdp4c Soybean Developing Pods(10-12 mm) sdp4c.pk006.n11:fis se4 Soybean Embryo, 19 Days AfterFlowering se4.cp0008.b2 sgs1c Soybean Seeds 4 Hours After Germinationsgs1c.pk001.i16 sgs1c Soybean Seeds 4 Hours After Germinationsgs1c.pk005.k7 sgs5c Soybean Seeds 4 Days After Germinationsgs5c.pk0003.e7 sl2 Soybean Two-Week-Old Developing Seedlings Treatedsl2.pk0033.c1 With 2.5 ppm chlorimuron sl2 Soybean Two-Week-OldDeveloping Seedlings Treated sl2.pk121.a19 With 2.5 ppm chlorimuronwle1n Wheat Leaf From 7 Day Old Etiolated Seedling¹ wle1n.pk0002.g3 wlk1Wheat Seedlings 1 Hour After Treatment With Herbicide⁵ wlk1.pk0004.e7wlm1 Wheat Seedlings 1 Hour After Inoculation With Erysiphewlm1.pk0018.g6 graminis f. sp tritici wre1n Wheat Root From 7 Day OldEtiolated Seedling¹ wre1n.pk0027.d4 wre1n Wheat Root From 7 Day OldEtiolated Seedling¹ wre1n.pk0040.h11 wre1n Wheat Root From 7 Day OldEtiolated Seedling¹ wre1n.pk0064.g7 ¹These libraries were normalizedessentially as described in U.S. Pat. No. 5,482,845, the disclosure ofwhich is hereby incorporated by reference. ²Chemicals used includedokadaic acid, cyclosporin A, calyculin A, cypermethrin. ³A23187 iscommercially available from several vendors including Calbiochem. ⁴Corndevelopmental stages are explained in the publication “How a corn plantdevelops” from the Iowa State University Coop. Ext. Service SpecialReport No. 48 reprinted June 1993. ⁵Application of6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods ofusing this compound are described in USSN 08/545,827, the disclosure ofwhich is hereby incorporated by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding LPAAT isozymes were identified by conducting BLAST(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol.215:403-410) searches for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Proteins Similar toMus musculus LPAAT

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the proteins encoded by the cDNAs to an unknownprotein from Caenorhabditis elegans and a putative LPAAT protein fromMus musculus (NCBI General Identifier Nos. 3878960 and 2317725,respectively). Shown in Table 3 are the BLAST results for individualESTs (“EST”) or for the sequences of contigs assembled from two or moreESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toMus musculus LPAAT BLAST pLog Score Clone Status 3878960 2317725 Contigof: Contig 59.40 57.70 p0018.chssd06r p0104.cabbd29r cca.pk0027.c9p0018.chstw94r p0094.csssl20r sl2.pk121.a19 EST 15.22 10.09 Contig of:Contig 54.30 50.52 wlm1.pk0018.g6 wre1n.pk0040.h11 wre1n.pk0064.g7

The sequence of the entire cDNA insert in clones p0018.chssd06r ands12.pk121.a19 was determined. Further sequencing and analysis of theDuPont proprietary EST database allowed the identification of catalpa,rice, and sorghum clones encoding polypeptides with similarities to Musmusculus LPAAT. The BLAST search using the sequences from clones listedin Table 4 revealed similarity of the proteins encoded by the cDNAs toan unknown protein from Caenorhabditis elegans and a putative LPAATprotein from Mus musculus (NCBI General Identifier Nos. 3878960 and2317725, respectively). Shown in Table 4 are the BLAST results for thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), the sequences of the entire protein encoded by a contigassembled from an FIS and one or more ESTs (“Contig*”), or the sequencesof the entire protein encoded by an FIS (“CGS”):

TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous toMus musculus LPAAT BLAST pLog Score Clone Status 3878960 2317725ncs.pk0013.d2:fis CGS 56.40 54.15 Contig of: Contig* 58.00 55.04ceb1.pk0011.d11 ceb5.pk0053.e3 p0010.cbpbq45r p0018.chssd06r:fisrlr2.pk0028.d6:fis CGS 57.70 55.40 gds1c.pk002.a19:fis FIS 58.10 45.52sl2.pk121.a19:fis CGS 57.70 53.00

In this type of plant LPAAT domain I consists of amino acidsAsn-His-Thr-Ser-Met-Ile-Asp-Phe-Ile and domain II (62 amino acidsdownstream) consists of amino acids Leu-Ile-Phe-Pro-Glu-Gly-Thr-Cys.

The data in Table 5 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4 6, 26, 28, 30, 32,and 34 and the Caenorhabditis elegans and Mus musculus sequences (NCBIGeneral Identifier Nos. 3878960 and 2317725, respectively).

TABLE 5 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toMus musculus LPAAT Percent Identity to SEQ ID NO. 3878960 2317725 2 38.535.1 4 39.3 29.9 6 39.8 35.9 26 31.8 35.4 28 32.1 36.1 30 31.9 37.4 3233.5 36.1 34 32.2 35.4

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a corn, a sorghum, a soybean and a wheatLPAAT and entire catalpa, corn, rice, and soybean LPAAT proteins. Thesesequences represent the first catalpa, corn, rice, soybean, and wheatsequences encoding LPAAT proteins of this type.

Example 4 Characterization of cDNA Clones Encoding LPAATs Similar toBurkholderia pseudomallei LPAAT

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to LPAATfrom Burkholderia pseudomallei (NCBI General Identifier No. 3135672).Shown in Table 6 are the BLAST results for individual ESTs (“EST”) thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), or for the sequences of contigs assembled from two ormore ESTs (“Contig”):

TABLE 6 BLAST Results for Sequences Encoding Polypeptides Homologous toBurkholderia pseudomallei LPAAT BLAST pLog Score Clone Status 3135672Contig of: Contig 9.52 ceb5.pk0049.b3 cen3n.pk0027.f6 sgs1c.pk001.i16FIS 9.30 wre1n.pk0027.d4 EST 4.00

The sequence of the entire cDNA insert from clone wre1n.pk0027.d4 wasdetermined. Further sequencing and analysis of the DuPont proprietarydatabase allowed the identification of a catalpa clone with similarityto the Burkholderia pseudomallei LPAAT. The BLAST search using thesequences from clones listed in Table 7 revealed similarity of thepolypeptides encoded by the Arabidopsis thaliana contig to similar toacyltransferase (NCBI General Identifier No. 6503307) and of the cDNAsto LPAAT from Burkholderia pseudomallei (NCBI General Identifier No.3135672). Shown in Table 7 are the BLAST results for the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones encodingthe entire protein (“CGS”):

TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous toBurkholderia pseudomallei LPAAT BLAST pLog Score Clone Status 65033073135672 ncs.pk0009.f12:fis CGS 87.00 10.22 wre1n.pk0027.d4:fis CGS 83.5211.40

In this type of plant LPAAT domain I consists of amino acidsAsn-His-(Val or Ile)-Ser-Tyr-(Val, Ile, or Leu)-Asp-Ile-Leu and domainII (62 amino acids downstream) consists of amino acids Xaa1-(Leu orIle)-Phe-Pro-Glu-Gly-Thr-Thr, where Xaa1 is Leu, Ile, Met or Tyr.

The data in Table 8 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:8, 10, 12, 36, and 38and the Burkholderia pseudomallei sequence (NCBI General Identifier No.3135672).

TABLE 8 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toBurkholderia pseudomallei LPAAT Percent Identity to SEQ ID NO. 3135672 819.8 10 17.6 12 17.4 36 219.1 38 20.3

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a wheat LPAAT and entire corn, catalpa,soybean, and wheat LPAAT proteins. These sequences represent the firstcorn, catalpa, soybean, and wheat sequences encoding LPAATs of thistype.

Example 5 Characterization of cDNA Clones Encoding Putative LPAATs

The BLASTX search using the EST sequences from clones listed in Table 9revealed similarity of the polypeptides encoded by the contig to anunknown protein from Arabidopsis thaliana (NCBI General Identifier No.2979560). Shown in Table 9 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or the sequences of contigs assembledfrom two or more ESTs (“Contig”):

TABLE 9 BLAST Results for Sequences Encoding Polypeptides Homologous toLPAATs BLAST pLog Score Clone Status 2979560 ads1c.pk005.i10 FIS 52.00Contig of: Contig 22.70 rls6.pk0076.d5 rlr24.pk0068.e3 scb1c.pk003.d18EST 45.04

The sequence of the entire cDNA insert in clones r1s6.pk0076.d5 andscb1c.pk003.d18 was determined. Further sequencing and analysis of theDuPont proprietary database allowed the identification of corn cloneswith similarities to the Arabidopsis thaliana putative protein. TheBLAST search using the sequences from clones listed in Table 10 revealedsimilarity of the polypeptides encoded by the contig to an unknownprotein from Arabidopsis thaliana (NCBI General Identifier No. 2979560).Shown in Table 10 are the BLAST results for the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”), the sequencesof contigs assembled from two or more ESTs (“Contig”), or the sequencesof an FIS encoding the entire protein (“CGS”):

TABLE 10 BLAST Results for Sequences Encoding Polypeptides Homologous toLPAATs BLAST pLog Score Clone Status 2979560 Contig of: Contig 21.70ceb1.mn0001.d12:fis cpe1c.pk006.e1 rls6.pk0076.d5:fis FIS 67.52scb1c.pk003.d18:fis CGS 81.00

In this type of plant LPAATs domain I includes the amino acidsSer-Asn-His-(Val or Ile)Ser-Tyr-Ile-Glu-Pro-Ile and domain II (61 aminoacids downstream) includes the amino acidsLeu-Leu-Phe-Pro-Glu-Gly-Thr-Thr-Thr.

The BLAST search using the sequences from clones listed in Table 11revealed similarity of the polypeptides encoded by the contig to amember of the acyltransferase family from Arabidopsis thaliana (NCBIGeneral Identifier No. 6503307). Shown in Table 11 are the BLAST resultsfor the sequences of the entire cDNA inserts comprising the indicatedcDNA clones (“FIS”), the sequences of contigs assembled from two or moreESTs (“Contig”), or the sequences of the entire protein encoded by anFIS (“CGS”):

TABLE 11 BLAST Results for Sequences Encoding Polypeptides Homologous toLPAATs BLAST pLog Score Clone Status 6503307 cco1n.pk062.p19:fis CGS119.00 rlr6.pk0094.f6:fis CGS 111.00 sdp4c.pk006.n11:fis FIS 95.52Contig of: Contig 6.52 sgs1c.pk005.k7 sgs5c.pk0003.e7

In this type of plant LPAATs domain I includes the amino acidsSer-Asn-His-Val-Ser-Tyr-(Val or Leu)-Asp-Ile-Leu and domain II (61 aminoacids downstream) includes the amino acidsLeu-Phe-Pro-Glu-Gly-Thr-Thr-Thr.

The data in Table 12 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:14, 16, 18, 40, 42, 44,46, 48, 50, and 52 and the Arabidopsis thaliana sequences (NCBI GeneralIdentifier No. 6503307).

TABLE 12 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toLPAATs Percent Identity to SEQ ID NO. 2979560 6503307 14 36.3 13.2 1632.8 13.8 18 65.4 16.8 40 27.0 21.1 42 50.2 16.9 44 65.4 19.7 46 18.054.6 48 18.1 52.5 50 11.2 63.7 52 12.4 19.5

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of an Arabidopsis, a corn, a rice, and asoybean LPAAT and an entire soybean LPAAT. These sequences represent thefirst corn, rice, soybean, and Arabidopsis sequences encoding LPAAT ofthis type.

Example 6 Characterization of cDNA Clones Encoding Proteins Similar toZea mays LPAAT

The BLASTX search using the EST sequences from clones listed in Table 13revealed similarity of the polypeptides encoded by the cDNAs to LPAATfrom Zea mays (NCBI General Identifier No. 575960). Shown in Table 13are the BLAST results for the sequences of contigs assembled from two ormore ESTs (“Contig”):

TABLE 13 BLAST Results for Sequences Encoding Polypeptides Homologous toZea mays LPAAT BLAST pLog Score Clone Status 575960 Contig of: Contig57.70 rr1.pk0004.a10 rr1.pk0039.e10 Contig of: Contig 67.15se4.cp0008.b2 sl2.pk0033.c1 Contig of: Contig 78.70 wlk1.pk0004.e7wle1n.pk0002.g3

The sequence of the entire cDNA insert in clones rr1.pk0004.a10,s12.pk0033.c1, and w1k1.pk0004.e7 was determined. The BLASTP searchusing the amino acid sequences from clones listed in Table 14 revealedsimilarity of the polypeptides encoded by the cDNAs to LPAATs from Zeamays and Brassica napus (NCBI General Identifier Nos. 1076821 and4583544, respectively). Shown in Table 14 are the BLAST results for thesequences of the entire cDNA inserts comprising the indicated cDNAclones encoding the entire protein (“CGS”):

TABLE 14 BLAST Results for Sequences Encoding Polypeptides Homologous toZea mays LPAAT BLAST pLog Score Clone Status 1076821 4583544rr1.pk0004.a10:fis CGS >254.00 149.00 sl2.pk0033.c1:fis CGS 169.00175.00 wlk1.pk0004.e7:fis CGS >254.00 148.00

In this type of plant LPAAT domain I consists of amino acidsSer-Asn-His-Arg-Ser-Asp-Ile-Asp-Trp-Leu and domain II (69 amino acidsdownstream) consists of amino acids Ala-Leu-Phe-Val-Glu-Gly-Thr-Arg-Phe.

The data in Table 15 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:20, 22, 24, 54, 56, and58 and the Zea mays sequence (NCBI General Identifier Nos. 1076821).

TABLE 15 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toZea mays LPAAT Percent Identity to SEQ ID NO. 1076821 20 72.6 22 72.4 2473.1 54 91.2 56 70.1 58 84.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of and an entire rice, soybean, and wheatLPAAT. These sequences represent the first rice, soybean, and wheatsequences encoding LPAATs of this type.

Example 7 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL 1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 8 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC 18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with fluorescent lights ona 16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 9 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Activity assays may be conducted under well known experimentalconditions which permit optimal enzymatic activity. For example, assaysfor LPAAT which incorporates medium-sized chain fatty acids arepresented by Knutzon et al. (1995) Plant Physiol. 109:999-1006. Assaysfor LPAAT which incorporates fatty acids longer than 18 carbons arepresented by Lassner et al. (1995) Plant Physiol. 109:1389-1394. Assaysto investigate the fatty acid selectivity of LPAATs is presented byLöhden and Frentzen (1992) Planta 188:215-224.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having lysophosphatidic acid acyltransferase(LPAAT) activity, wherein the polypeptide has an amino acid sequence ofat least 95% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:34; or (b) a complement of thenucleotide sequence, wherein the complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary. 2.The polynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO:34.
 3. The polynucleotide of claim 1wherein the nucleotide sequence comprises SEQ ID NO:33.
 4. A vectorcomprising the polynucleotide of claim
 1. 5. A recombinant DNA constructcomprising the polynucleotide of claim 1 operable linked to at least oneregulatory sequence.
 6. A method for transforming a cell, comprisingtransforming a cell with the polynucleotide of claim
 1. 7. A cellcomprising the recombinant DNA construct of claim
 5. 8. A method forproducing a plant comprising transforming a plant cell with thepolynucleotide of claim 1 and regenerating a plant from the transformedplant cell.
 9. A plant comprising the recombinant DNA construct of claim5.
 10. A seed comprising the recombinant DNA construct of claim
 5. 11. Amethod for isolating a polypeptide comprising isolating the polypeptidefrom a cell or culture medium of the cell, wherein the cell comprises arecombinant DNA construct comprising the polynucleotide of claim 1operable linked to at least one regulatory sequence.
 12. A method ofaltering the level of lysophosphatidic acid acyltransferase expressionin a host cell comprising: (a) transforming a host cell with therecombinant DNA construct of claim 5; and (b) growing the transformedhost cell under conditions that are suitable for expression of therecombinant DNA construct wherein expression of the recombinant DNAconstruct results in production of altered levels of lysophosphatidicacid acyltransferase in the transformed host cell.